Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors regulating many key metabolic pathways. Much of the insight into PPARs derives from studies with synthetic PPAR agonists, including those in clinical use. PPARa, activated by lipid-lowering fibrates, regulates fatty acid metabolism. PPARy, activated by insulin-sensitizing thiazolidinediones, controls adipogenesis and glucose homeostasis. Both PPARa and PPARy activation may limit atherosclerosis and inflammation. This body of data for PPAR activation by synthetic agonists establishes the importance of understanding endogenous PPAR activation. Recently, we reported lipoprotein lipase (LPL) acts on circulating lipoproteins in a specific and selective manner to generate PPARalpha ligands. This proposal focuses on the central hypothesis that direct and indirect pathways for endogenous PPAR antagonism play an important part in determining metabolic responses. Data is provided for three different endogenous pathways that may negatively regulate PPAR activity. These mechanisms will be studied by examining their modulation of well established in vitro and in vivo models of PPAR activation. LPL-mediated PPARa activation suggests the discrepant endothelial effects of structurally diverse fatty acids may be due to differential PPARa activation, including antagonism. In Aim 1, PPAR activation and inhibition by specific fatty acids will be studied in vitro and in vivo, contrasting omega-3 fatty acids to saturated and trans-fatty acids. Hepatic nuclear factor 4 alpha (HNF4alpha) is a poorly understood but critical fatty acidactivated receptor that regulates lipid metabolism, thrombosis, and glucose control. By using the manipulations of lipid metabolism employed in our LPL/PPARa studies, we have identified novel mechanisms of HNF4alpha modulation and divergent responses between PPARalpha and HNF4alpha to pathways of lipid metabolism. In Aim 2, this divergence between HNF4alpha and PPARalpha will be explored. A novel but powerful mechanism for PPAR modulation would be the existence of a direct endogenous PPAR antagonist. While symmetric cleavage of beta carotene generates natural ligands for the RXR nuclear receptor, we have identified that asymmetric beta carotene cleavage produces a specific apocarotenal that directly antagonizes PPAR responses. In Aim 3, this direct antagonist will be characterized in vitro and in vivo. Together, these studies integrate biochemical, biologic, and in vivo models to better understand how endogenous modulation of PPAR activity may determine biologic responses.